Jane Kasten-Jolly

Folding Domains within the Ricin Toxin A Subunit as Targets of Protective Antibodies

Efforts to develop an effective vaccine against ricin are focused on the engineering of attenuated and stable recombinant forms of the toxins enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of those regions of RTA that are critical in eliciting protective immunity. In this present study, we produced and characterized a collection of monoclonal antibodies (MAbs) directed against five distinct immunodominant regions on RTA, and used these MAbs to identify several key neutralizing epitopes on the toxin. Protective MAbs were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.

Susceptibility of Lupus-Prone Nzm Mouse Strains to Lead Exacerbation of Systemic Lupus Erythematosus Symptoms

It has been repeatedly shown that the heavy metal mercury can induce or exacerbate lupuslike autoimmunity in susceptible strains of rats and mice. A hallmark of such autoimmune induction is the accompaniment of an immune shift, in which there is usually an initial skewing toward a Th2-like immune environment. Another heavy metal, lead (Pb), has also been found to induce a Th2 shift in mice. However, exposure of normal mouse strains to Pb does not appear to induce autoimmunity. In order to investigate whether mice genetically predisposed to murine systemic lupus erythematosus (SLE) are susceptible to a Pb-induced exacerbation of lupus, males and females of four New Zealand mixed (NZM) mouse strains, along with BALB/c and C57Bl/6 controls, were administered three 100-µl intraperitoneal injections of either 1.31 mM lead or sodium acetate per week for 3 wk. The four NZM strains chosen, NZM391, NZM2328, NZM88, and NZM2758, have differential genetic penetrance for SLE with variances in certain manifestations of the disease, but all of these strains naturally develop glomerulonephritis and produce high titers of anti-nuclear autoantibodies. The mice were prebled for baseline values and were bled directly after the injection period (d 1) and monthly thereafter for 5 mo. Sera were assessed for anti-double-stranded DNA titers, urea nitrogen levels, and creatine kinase activity, as well as for total immunoglobulin (Ig) G2a and IgG1 levels. Mortality and morbidity of the mice were also recorded. All NZM strains showed an acute, non-gender-based, susceptibility to Pb at d 1, but the control strains were unaffected. Over time, it became apparent that the strains diverged: The NZM391 strain showed gender-independent susceptibility to Pb enhancement of lupus manifestations and mortality; the NZM2328 strain exhibited gender-independent Pb susceptibility to manifestations, although only females had increased mortality; the NZM2758 strain exhibited non-gender-based elevations in urea nitrogen and creatine kinase activity levels; and the NZM88 strain displayed male susceptibility to anti-DNA and life span. Surprisingly, Pb increased the longevity of NZM88 and NZM2758 females. These results indicate that Pb indeed can exacerbate SLE in lupus-prone mice; however, even among lupus-prone strains, genetic differences determine the degree of exacerbation. Using the known phenotype and genetic differences, one can identify and characterize possible traits and loci associated with Pb susceptibility.

Developmental lead effects on behavior and brain gene expression in male and female BALB/cAnNTac mice

Lead (Pb) was one of the first poisons identified, and the developing nervous system is particularly vulnerable to its toxic effects. Relatively low, subclinical doses, of Pb that produce no overt signs of encephalopathy can affect cognitive, emotional, and motor functions. In the present study, the effects of developmental Pb-exposure on behavioral performance and gene expression in BALB/cAnNTac mice were evaluated. Pups were exposed to Pb from gestational-day (gd) 8 to postnatal-day (pnd) 21 and later evaluated in exploratory behavior, rotarod, Morris water maze, and resident-intruder assays as adults. Pb-exposure caused significant alterations in exploratory behavior and water maze performance during the probe trial, but rotarod performance was not affected. Pb-exposed males displayed violent behavior towards their cage mates, but not to a stranger in the resident-intruder assay. Gene expression analysis at pnd21 by microarray and qRT-PCR was performed to provide a molecular link to the behavior changes that were observed. Pb strongly up-regulated gene expression within the signaling pathways of mitogen activated protein kinases (MAPKs), extra-cellular matrix (ECM) receptor, focal adhesion, and vascular endothelial growth-factor (VEGF), but Pb down-regulated gene expression within the pathways for glycan structures-biosynthesis 1, purine metabolism, and N-glycan biosynthesis. Pb increased transcription of genes for major histocompatibility (MHC) proteins, the chemokine Ccl28, chemokine receptors, IL-7, IL7R, and proteases. The qRT-PCR analysis indicated an increase of gene expression in the whole brain for caspase 1 and NOS2. Analysis of IL-1β, caspase 1, NOS2, Trail, IL-18 and IL-33 gene expression of brain regions indicated that Pb perturbed the inter-regional expression pattern of pro-inflammatory genes. Brain region protein concentrations for IL-10, an anti-inflammatory cytokine, showed a significant decrease only within the cortex region. Results indicate that Pb differentially affects the behavior of male and female mice in that females did less exploration and the males were selectively more aggressive. Gene expression data pointed to evidence of neuroinflammation in the brain of both female and male mice. Pb had more of an effect in the males on expression of vomeronasal receptor genes associated with odor detection and social behavior.

Lead Modulation of Macrophages Causes Multiorgan Detrimental Health Effects

The environmental toxicant lead (Pb) has detrimental effects on a number of organ systems, including the immune system. Pb exposure decreases host immune defenses against numerous microorganisms and cancer. Although Pb effects on humoral and cell‐mediated immunity as well as on erythrocyte, neural, and renal pathophysiology have been well documented, there are few reports regarding Pbs impact on innate immunity, which can affect multiorgan processes. This review focuses on Pb modulation of a key innate immune cell, the macrophage. The impact of Pb on macrophages in different organs, on immature versus mature macrophages, and on low versus high Pb concentrations is discussed. Pb decreases phagocytosis and chemotaxis of macrophages and affects nitric oxide production and eicosanoid metabolism in mature macrophages. Pretreatment of macrophages with Pb increases TNF‐α secretion after in vitro stimulation with lipopolysaccharide; however, Pb exposure decreases in vivo intracellular pathogen killing. More recent evidence from mouse studies indicates that even low, environmentally relevant, blood concentrations of Pb result in increased phagocytosis of erythrocytes and decreased expression of interferon‐gamma‐inducible GTPases, p65‐GBP, and p47‐IRG, which are necessary for intracellular pathogen killing. Taking into account the effects of Pb on macrophages, the review describes posited mechanisms to account for Pb‐altered health effects; Pb effects on heme levels may play a key role as well as Pbs preferential induction of helper type‐2 T (Th2) cells and M2 macrophages, which is related to oxidative stress. The discussion links old findings with new, thereby adding new insight into the effects of Pb on macrophages and the resultant compromised immunity and health.

Metallothionein differentially affects the host response to Listeria infection both with and without an additional stress from cold-restraint

Acute stress alters anti-bacterial defenses, but the neuroimmunological mechanisms underlying this association are not yet well understood. Metallothionein (MT), a cysteine-rich protein, is a stress response protein that is induced by a variety of chemical, biological, and psychological stressors, and MT has been shown to influence immune activities. We investigated MT’s role in the management of anti-bacterial responses that occur during stress, using a C57BL/6 (B6) strain that has targeted disruptions of the Mt1 and Mt2 genes (B6-MTKO), and a B6 strain that has additional copies of Mt (B6-MTTGN). The well-characterized listeriosis model was used to examine immune mechanisms that are altered by a 1-h stress treatment (cold-restraint, CR) administered just prior to bacterial infection. Intriguingly, MT gene doses both greater and lower than that of wild-type (WT) B6 mice were associated with improved host defenses against Listeria monocytogenes (LM). This augmented protection was diminished by CR stress in the MTKO mice, but transgenic mice with additional MT copies had no CR stress-induced increase in their listerial burden. During the transition from innate to adaptive immunity, on day 3 after infection, oxidative burst and apoptosis were assessed by flow cytometric methods, and cytokine transcription was measured by real-time quantitative PCR. MT gene expression and CR-stress affected the expression of IL-6 and TNFα. Additionally, these genetic and environmental modulations altered the generation of ROS responses as well as the number of apoptotic cells in livers and spleens. Although the level of MT altered the listerial response, MT expression was equally elevated by listerial infection with or without CR stress. These results indicate the ability of MT to regulate immune response mechanisms and demonstrate that increased amounts of MT can eliminate the immunosuppression induced by CR.

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2h, host defenses against bacterial growth are suppressed 2-3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidation (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes.